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Development：北京林業大學丨固醇調控FLS2蛋白胞吞的新機制
NMT是基因功能的活體檢測技術，已被31位諾貝爾獎得主所在單位，及北大、清華、中科院使用。
期刊：Development
主題：固醇調控FLS2蛋白胞吞的新機制（鈣信號）
標題：Sterols regulate endocytic pathways during flg22-induced defense responses in Arabidopsis
影響因子：5.413
檢測指標：Ca2+流速
檢測樣品：擬南芥葉片
Ca2+流速流實驗處理方法：
7日齡擬南芥，0.1 μM flg22處理
Ca2+流速流實驗測試液成份：無
推薦測試液：0.1mM CaCl2，pH 6.0
作者：北京林業大學林金星、李曉娟
英文摘要
The plant transmembrane receptor kinase FLAGELLIN SENSING 2 (FLS2) is crucial for innate immunity. Although previous studies have reported FLS2-mediated signal transduction and endocytosis via the clathrin-mediated pathway, whether additional endocytic pathways affect FLS2-mediated defense responses remains unclear.
Here, we show that the Arabidopsis thaliana sterol-deficient mutant steroid methyltransferase 1 displays defects in immune responses induced by the flagellin-derived peptide flg22. Variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) coupled with single-particle tracking showed that the spatiotemporal dynamics of FLS2-GFP changed on a millisecond time scale and that the FLS2-GFP dwell time at the plasma membrane increased in cells treated with a sterol-extracting reagent when compared with untreated counterparts.
We further demonstrate that flg22-induced FLS2 clustering and endocytosis involves the sterol-associated endocytic pathway, which is distinct from the clathrin-mediated pathway. Moreover, flg22 enhanced the colocalization of FLS2-GFP with the membrane microdomain marker Flot 1-mCherry and FLS2 endocytosis via the sterol-associated pathway. This indicates that plants may respond to pathogen attacks by regulating two different endocytic pathways.
Taken together, our results suggest the key role of sterol homeostasis in flg22-induced plant defense responses.
中文摘要（谷歌機翻）
植物跨膜受體激酶FLAGELLIN SENSING 2（FLS2）對于先天免疫至關重要。盡管以前的研究已經報道了通過網格蛋白介導的途徑介導的FLS2介導的信號轉導和胞吞作用，但尚不清楚其他內吞途徑是否會影響FLS2介導的防御反應。
在這里，我們顯示擬南芥的甾醇缺乏突變體類固醇甲基轉移酶1在鞭毛蛋白衍生的肽flg22誘導的免疫反應中顯示缺陷。可變角度全內反射熒光顯微鏡（VA-TIRFM）結合單粒子跟蹤顯示，在處理的細胞中，FLS2-GFP的時空動態在毫秒級變化，質膜上的FLS2-GFP停留時間增加與未經處理的對應物相比，含固醇提取劑。
我們進一步證明，flg22誘導的FLS2聚集和內吞涉及與固醇相關的內吞途徑，這不同于網格蛋白介導的途徑。此外，flg22通過固醇相關途徑增強了FLS2-GFP與膜微區標記Flot 1-mCherry和FLS2內吞的共定位。這表明植物可以通過調節兩種不同的內吞途徑來響應病原體侵襲。
兩者合計，我們的結果表明固醇穩態在flg22誘導的植物防御反應中的關鍵作用。
結果表明：相比野生型，smt1（甾醇甲基轉移酶1）突變體表現出迅速且強烈的Ca2+吸收速率高于根冠（R0和R1），低于根伸長區（R2）。增加，說明突變體對于flg22更為敏感。smt1突變沒有改變FLS2的同源寡聚狀態，但影響FLS2簇形成。smt1突變體中FLS2的內吞功能受到損傷。由上述結果得到一個假設，即甾醇相關的內吞途徑對于flg22誘導的FLS2動力學和植物防御至關重要。